Ornithin Decarboxylase (ODC)
Polyamines are basic aliphatic hydrocarbon compounds with two or more amino groups. Polyamines are ubiquitous natural substances occurring in organism with more than 20 types described. Examples of polyamines are spermine, spermidine and putrescine. Biological roles attributed to polyamines in plants include cell protection during abiotic stress and promotion of nucleic acid or protein biosynthesis. Enzymes catalyzing essential steps in polyamine biosynthesis include Spermine Synthase, Spermidine Synthase (SPDS) and several basic amino acid decarboxylases of the beta/alpha barrel fold type such as Ornithine Decarboxylases (ODC), Arginine Decarboxylase (ADC), S-adenosylmethionine Decarboxylase (SAMDC), Diaminopimelate Decarboxylase (DAPCD) and Carboxynorspermidine Decarboxylase (CANSDC). Genes encoding such enzymes have been isolated from prokaryotic and eukaryotic organism including plants. Beta/alpha-barrel fold decarboxylases segregate with respect to their phylogeny into four distinct groups containing ADCs, DAPDCs, ODCs, and CANSDCs (Lee et al. 2007. The Journal of Biological Chemistry Vol. 282, 27115-27125). These enzymes form homodimers. Two identical active sites are formed at the dimer interface between the N-terminal domain from one subunit and the C-terminal domain from the other.
Ornithine Decarboxylase or L-ornithine carboxy-lyase catalyses the decarboxilation of L-Ornithine to produce putrescine and CO2. ODCs are found in prokaryotes and eukaryotic organisms. The nomenclature assigned to ODC by the International Union Of Biochemistry And Molecular Biology is EC 4.1.1.17.
There has been considerable research in understanding the expression of plant polyamine metabolism-relate genes in response to stresses, and the use of such genes to alter the polyamine concentration in a cell. For example expression of a mouse ODC in carrot or a human ODC in transgenic rice plants altered the polyamine pools in the transgenic plant (Bastola and Minocha 1995. Plant Physiol. 1995. 109(1): 63-71. Lepri et al; 2001 Mol Genet Genomics. 2001 October; 266(2):303-12.). Immunomodulation of ODC in tobacco plants reportedly resulted in altered polyamine levels and developmental abnormalities and drawf phenotypes in the transgenic plants (Nolke et al; 2005. plant Biotechnology J. 3(2): 237-47. Expression of a cDNA encoding a mouse ODC in tobacco increased putrescine levels (Descenzo and Minocha 1993) Plant Mol Biol. 22, 113-127. In this study most transformant plants had normal appearance although those accumulating high levels of putrescine displayed stunted growth, wrinkled leaves and flowers with reduced stamen.
Surprisingly, it has now been found that modulating expression of a nucleic acid encoding an Ornithine Decarboxylase polypeptide gives plants having enhanced yield-related traits relative to control plants.
According to one embodiment, there is provided a method for enhancing yield related traits of a plant relative to control plants, comprising modulating expression of a nucleic acid encoding an Ornithine Decarboxylase polypeptide in a plant. The enhanced yield related traits comprised increased early vigour, increased seed yield and increased biomass.
Benzothiadiazole-induced Homeodomain Protein 1 (BIHD1)
DNA-binding proteins are proteins that comprise any of many DNA-binding domains and thus have a specific or general affinity to DNA. DNA-binding proteins include for example transcription factors that modulate the process of transcription, nucleases that cleave DNA molecules, and histones that are involved in DNA packaging in the cell nucleus.
Transcription factors are usually defined as proteins that show sequence-specific DNA binding affinity and that are capable of activating and/or repressing transcription. The Arabidopsis thaliana genome codes for at least 1533 transcriptional regulators, accounting for ˜5.9% of its estimated total number of genes (Riechmann et al. (2000) Science 290: 2105-2109). The Database of Rice Transcription Factors (DRTF) is a collection of known and predicted transcription factors of Oryza sativa L. ssp. indica and Oryza sativa L. ssp. japonica, and currently contains 2,025 putative transcription factors (TF) gene models in indica and 2,384 in japonica, distributed in 63 families (Gao et al. (2006) Bioinformatics 2006, 22(10):1286-7).
One of these families is the superfamily of homeodomain (HD) transcription factors involved in many aspects of developmental processes. HD transcription factors are characterized by the presence of a homeodomain (HD), which is a 60-amino acid DNA-binding domain (BD). Arabidopsis thaliana and rice contain approximately 100 HD transcription factors, which can be further classified into subfamilies based on amino acid sequence identity (Richardt et al. (2007) Plant Phys 143(4): 1452-1466). Some of these subfamilies are characterized by the presence of additional conserved domains that facilitate DNA binding and/or protein-protein interactions.
One of these subfamilies is the BEL1 (BELL-1) subfamily (Reiser et al. (1995), Cell 83: 735-742), named after an Arabidopsis mutant bell1 with defective integument formation. BEL1 transcription factors are characterized, in addition to the homeodomain, by a conserved domain called the POX domain, found exclusively in plant proteins. Two motifs further characterize BEL1 polypeptides: the SKY box, and the VSLTGL box, named after the conserved amino acid residues they comprise.
A gene encoding a BEL1 homeodomain polypeptide was isolated from Oryza sativa, and its expression shown to be increased upon treatment with benzothiadiazole (BTH), a molecule capable of inducing disease resistance, but also upon inoculation with Magnaporthe grisea pathogen (Luo et al. (2005) Plant Biol 7: 459-468). It was named Oryza sativa benzothiadiazole induced homeodomain 1 (OsBIHD1). The gene was overexpressed in tobacco using the CaMV 35S promoter (Luo et al. (2005) J Ex Bot 56(420): 2673-2682). Transgenic tobacco plants showed enhanced disease tolerance, and in some instance, germinated apical buds, abnormal roots, reduced fertility or infertility.
Surprisingly, it has now been found that increasing expression of a nucleic acid sequence encoding a benzothiadiazole-induced homeodomain protein 1 (BIHD1) gives plants having increased yield-related traits relative to control plants.
According to one embodiment, there is provided a method for increasing yield-related traits in plants relative to control plants, comprising increasing expression of a nucleic acid sequence encoding a BIHD1 polypeptide as defined herein, in a plant. The increased yield-related traits comprise one or more of: increased early vigour, increased total seed yield per plant, increased number of filled seeds, increased total number of seeds, and increased harvest index.
MYB30 Protein
MYB domain proteins are transcription factors with a highly conserved DNA-binding domain. The MYB domain was originally described in the oncogene (v-myb) of avian myeloblastosis virus (Klempnauer et al. (1982) Cell 33, 453-63). Many vertebrates contain three genes related to v-Myb, c-Myb, A-Myb and B-Myb and other similar genes have been identified in insects, plants, fungi and slime moulds. The encoded proteins are crucial to the control of proliferation and differentiation in a number of cell types. MYB proteins contain one to four imperfect direct repeats of a conserved sequence of 50-53 amino acids which encodes a helix-turn-helix structure involved in DNA binding (Rosinski and Atchley (1998) J Mol Evol 46, 74-83). Three regularly spaced tryptophan residues, which form a tryptophan cluster in the three-dimensional helix-turn-helix structure, are characteristic of a MYB repeat. The three repeats in c-Myb are referred to as R1, R2 and R3; and repeats from other MYB proteins are categorised according to their similarity to R1, R2 or R3. MYB proteins can be classified into three subfamilies depending on the number of adjacent repeats in the MYB domain (one “MYB1R”, two “R2R3-type MYB”, three “MYB3R”). Since there is little sequence conservation outside of the MYB domain, MYB proteins have been clustered into subgroups based on conserved motifs identified outside of the MYB coding region (Stracke et al. 2001. Curr Opin Plant Biol. October; 4(5):447-56; Jiang et al. (2004) Genome Biology 5, R46). In contrast to animals, plants contain a MYB-protein subfamily that is characterised by the R2R3-type MYB domain.
Plant Myb genes are have been suggested to play important roles in regulation of secondary metabolism, cellular morphogenesis, pathogen resistance, and responses to growth regulators and stress. Additionally WO 2007099096 discloses a rice MYB4 protein useful for increasing seed yield in plants.
The MYB30 class of transcription factors constitutes a subgroup of Myb proteins sharing a common evolutionary origin and corresponding to the Group G09 reported by Jiang et al. 2004. Genes encoding some members of the MYB30 class of proteins have been implicated in physiological responses in guard cells and activation of the hypersensitive cell death response in Arabidopsis thaliana (Cominelli et al. Curr Biol. 2005 15(13):1196-200; Rivas and Roby FEBS Lett. 2006. 580(14):3498-504). The accumulation of extracellular VLCFA (very-long-chain fatty acids)-derived metabolites (leaf epidermal wax components) was affected in MYB30 knockout mutants and overexpressing lines in Arabidopsis thaliana (Vailleau et al. 2008. Plant Cell. March 7 [Epub ahead of print].).
Surprisingly, it has now been found that modulating expression of a nucleic acid encoding a MYB30 polypeptide gives plants having enhanced yield-related traits, in particular increased vegetative biomass and increased emergence vigour relative to control plants.
According to one embodiment, there is provided a method for enhanced yield-related traits of a plant relative to control plants, comprising modulating expression of a nucleic acid encoding a MYB30 polypeptide in a plant. The improved yield-related traits comprise increased biomass and increased emergence vigour.
Tomato Homeodomain (THOM)
Homeodomain leucine zipper (HDZip) proteins constitute a family of transcription factors characterized by the presence of a DNA-binding domain (HD) and an adjacent leucine zipper (Zip) motif. The homeodomain usually consists of 60 conserved amino acid residues that form a helix1-loop-helix2-turn-helix3 that binds DNA. This DNA binding site is usually pseudopalindromic. The leucine zipper, adjacent to the C-terminal end of the homeodomain, consists of several heptad repeats (at least four) in which usually a leucine (occasionally a valine or an isoleucine) appears every seventh amino acid. The leucine zipper is important for protein dimerisation. This dimerisation is a prerequisite for DNA binding (Sessa et al. (1993) EMBO J 12(9): 3507-3517), and may proceed between two identical HDZip proteins (homodimer) or between two different HDZip proteins (heterodimer).
Homeodomain genes are present in all eucaryotes, and constitute a gene family of at least 89 members in Arabidopsis thaliana. The leucine zipper is also found by itself in eukaryotes other than plants. However, the presence of both a homeodomain and a leucine zipper is plant-specific (found in at least 47 out of the 89 proteins in Arabidopsis), and has been encountered in moss in addition to vascular plants (Sakakibara et al. (2001) Mol Biol Evol 18(4): 491-502, which is incorporated herein by reference). The leucine zipper is then located at the C-terminal end of the homeodomain, these two features overlapping by three amino acids.
The Arabidopsis HDZip genes have been classified into four different classes, HDZip I to IV, based on sequence similarity criteria (Sessa et al. In: Plant Molecular Biology (NATO ASI Series, vol H81), pp 412-426, 1994). HD-Zip I and II genes are likely involved in signal transduction networks of light, dehydration-induced ABA, or auxin. These signal transduction networks are related to the general growth regulation of plants. The overexpression of sense or antisense HD-Zip I or II mRNA usually alters growth rate and development. Most members of the HD-Zip III subfamily play roles in cell differentiation in the stele. HD-Zip IV genes are related to the differentiation of the outermost cell layer (Sakakibara et al., 2001).
Several members of the closely related HD-Zip I and II families have been related to auxin signaling and transport. HDZip I and II genes also have been implicated in light signalling responses, including shade-avoidance, de-etiolation of dark-grown seedlings and blue light signaling. Furthermore, there is accumulating evidence that many HD-Zip I and II genes are related to regulation of developmental adaptation to environmental stress conditions such as drought, for an overview, see Agalou et al., Plant Molecular Biology 66, 87-103, 2008. By random binding site selection in vitro the favoured recognition site for the Arabidopsis HD-Zip family I protein Athb-1 was shown to be composed of two 5-bp half-sites that overlap at a central position, CAAT(A/T)ATTG (Sessa et al. EMBO J. 12, 3507-3517, 1993). The HD-Zip II protein Athb-2 interacts with a similar 9-bp sequence but shows a preference for a G/C base pair at the central position. This preference was shown to be determined by the presence of Glu and Thr residues at positions 46 and 56 of the 60-amino acid homeodomain, where HD-Zip I proteins characteristically contain, respectively, an Ala and a Trp residue (Sessa et al. J. Mol. Biol. 274, 303-309, 1997). THOM1 from tomato (Meisner and Theres, Planta 195, 541-547, 1995) is highly expressed in the vegetative shoot apical meristem, the floral meristem and axillary meristems. Young derivatives of these meristems show similar levels of THOM1 transcripts which decrease with increasing age of the respective tissue. HB-4, a member of the HD-Zip class II proteins and homologous to THOM, was shown to be induced in Arabidopsis by far-red-rich light treatment; whereas in sunflower, HB-4 is regulated by water availability and abscisic acid. It was postulated that sunflower HB-4 is involved in increasing desiccation tolerance (Manavella et al., Plant J. 48, 125-137, 2006).
Surprisingly, it has now been found that modulating expression of a nucleic acid encoding a THOM polypeptide gives plants having enhanced yield-related traits, in particular increased seed yield relative to control plants.
According one embodiment, there is provided a method for improving yield related traits of a plant relative to control plants, comprising modulating expression of a nucleic acid encoding a THOM polypeptide in a plant.
Benzothiadiazole-induced Homeodomain Protein 2 (BIHD2)
DNA-binding proteins are proteins that comprise any of many DNA-binding domains and thus have a specific or general affinity to DNA. DNA-binding proteins include for example transcription factors that modulate the process of transcription, nucleases that cleave DNA molecules, and histones that are involved in DNA packaging in the cell nucleus.
Transcription factors are usually defined as proteins that show sequence-specific DNA binding affinity and that are capable of activating and/or repressing transcription. The Arabidopsis thaliana genome codes for at least 1533 transcriptional regulators, accounting for ˜5.9% of its estimated total number of genes (Riechmann et al. (2000) Science 290: 2105-2109). The Database of Rice Transcription Factors (DRTF) is a collection of known and predicted transcription factors of Oryza sativa L. ssp. indica and Oryza sativa L. ssp. japonica, and currently contains 2,025 putative transcription factors (TF) gene models in indica and 2,384 in japonica, distributed in 63 families (Gao et al. (2006) Bioinformatics 2006, 22(10):1286-7).
One of these families is the superfamily of homeodomain (HD) transcription factors involved in many aspects of developmental processes. HD transcription factors are characterized by the presence of a homeodomain (HD), which is a 60-amino acid DNA-binding domain (BD). Arabidopsis thaliana and rice contain approximately 100 HD transcription factors, which can be further classified into subfamilies based on amino acid sequence identity (Richardt et al. (2007) Plant Phys 143(4): 1452-1466). Some of these subfamilies are characterized by the presence of additional conserved domains that facilitate DNA binding and/or protein-protein interactions.
One of these subfamilies is the BEL1 (BELL-1) subfamily (Reiser et al. (1995), Cell 83: 735-742), named after an Arabidopsis mutant bell1 with defective integument formation. BEL1 transcription factors are characterized, in addition to the homeodomain, by a conserved domain called the POX domain, found exclusively in plant proteins. Two motifs further characterize BEL1 polypeptides: the SKY box, and the VSLTGL box, named after the conserved amino acid residues they comprise.
A gene encoding a BEL1 homeodomain polypeptide was isolated from Oryza sativa, and its expression shown to be increased upon treatment with benzothiadiazole (BTH), a molecule capable of inducing disease resistance, but also upon inoculation with Magnaporthe grisea pathogen (Luo et al. (2005) Plant Biol 7: 459-468). It was named Oryza sativa benzothiadiazole induced homeodomain 1 (OsBIHD1). The gene was overexpressed in tobacco using the CaMV 35S promoter (Luo et al. (2005) J Ex Bot 56(420): 2673-2682). Transgenic tobacco plants showed enhanced disease tolerance, and in some instance, germinated apical buds, abnormal roots, reduced fertility or infertility.
Surprisingly, it has now been found that increasing expression of a nucleic acid sequence encoding a benzothiadiazole-induced homeodomain protein 2 (BIHD2) gives plants having increased yield-related traits relative to control plants.
According to one embodiment, there is provided a method for increasing yield-related traits in plants relative to control plants, comprising increasing expression of a nucleic acid sequence encoding a BIHD2 polypeptide as defined herein, in a plant. The increased yield-related traits comprise one or more of: increased early vigour, increased total seed yield per plant, increased number of filled seeds, increased total number of seeds, and increased harvest index.